Disassembly of the ring‐type decameric structure of peroxiredoxin from Aeropyrum pernix K1 by amino acid mutation

Abstract

The quaternary structure of peroxiredoxin from Aeropyrum pernix K1 (ApPrx) is a decamer, in which five homodimers are assembled in a pentagonal ring through hydrophobic interactions. In this study, we determined the amino acid (AA) residues of ApPrx crucial for forming the decamer using AA mutations. The ApPrx0Cys mutant, wherein all cysteine residues were mutated to serine, was prepared as a model protein to remove the influence of the redox states of the cysteines on its assembling behavior. The boundary between each homodimer of ApPrx0Cys contains characteristic aromatic AA residues forming hydrophobic interactions: F46, F80, W88, W210, and W211. We found that a single mutation of F46, F80, or W210 to alanine completely disassembled the ApPrx0Cys decamer to homodimers, which was clarified by gel-filtration chromatography and dynamic light scattering measurements. F46A, F80A, and W210A mutants lacked only one aromatic ring compared with ApPrx0Cys, indicating that the assembly is very sensitive to the surface structure of the protein. X-ray structures revealed two mechanisms of disassembly of the ApPrx decamer: loss of hydrophobicity between homodimers and flip of the arm domain. The AA residues targeted in this study are well conserved in ring-type Prx proteins, suggesting the importance of these residues in the assembly. This study demonstrates the sensitivity and modifiability of peroxiredoxin assembly by a simple AA mutation. K E Y W O R D Salanine substitution, hydrophobic interaction, peroxiredoxin, protein assembly

Rebuilding Ring-Type Assembly of Peroxiredoxin by Chemical Modification

“…Finally, reconstitution of dodecameric assembly was achieved through mutation to the building block ApPrx*F80C protein. As we reported previously, ApPrx*K84A mutant forms hexagonal dodecamer, in which the 84th AA residue located in the boundary of dimers is mutated to Ala to modify the interface interaction . Afterward, we determined its crystal structure at 2.9 Å resolution, confirming the hexagonal assembly (Figure S12).…”

“…Gel-filtration chromatography and dynamic light scattering (DLS) revealed that the ApPrx*F80C mutant in solution assumed the dimeric state. Gel-filtration chromatography revealed that ApPrx*F80C eluted at 15.1 mL, which is the equivalent elution volume of an ApPrx*F80A mutant assuming a dimeric state (Figure S2a, Figure a) . The hydrodynamic diameter measured by DLS was 4.85–5.61 nm at a protein concentration of 1.3–20 g/L, which corresponded to the size of dimeric ApPrx*F80A (Figure S2b).…”

“…ApPrx*F80C assembled a decamer-like crystal form although it was dimeric in solution, suggesting that ApPrx*F80C packed into the decameric structure, similar to the native form in crystal but does not assemble in solution because of the lack of interaction among dimers, as previously observed for the ApPrx*F80A mutant. 30 The crystal structures of Ph@ApPrx* and Naph@ApPrx* also represented ring-type decamers similar to native ApPrx. The Ph and Naph moieties were linked on C80.…”

“…A mutation causing changes in any of these residues to alanine (Ala) disassembled ApPrx* to dimers owing to the absence of hydrophobic interactions among the dimers. 30 The elimination of a single aromatic moiety per monomer disassembled decameric ApPrx* to dimers. This inspired us to reconstitute its QS through chemical modifications, wherein a synthetic aromatic ring is added to a protein surface to recover proteinprotein interactions.…”

“…Amino acid residues F46, F80, and W210 are important in maintaining the decameric assembly. A mutation causing changes in any of these residues to alanine (Ala) disassembled ApPrx* to dimers owing to the absence of hydrophobic interactions among the dimers . The elimination of a single aromatic moiety per monomer disassembled decameric ApPrx* to dimers.…”